Gluconeogenesis in the extraembryonic yolk syncytial layer of the zebrafish embryo.

Yolk-consuming (lecithotrophic) embryos of oviparous animals, such as those of fish, need to make do with the maternally derived yolk. However, in many cases, yolk possesses little carbohydrates and sugars, including glucose, the essential monosaccharide. Interestingly, increases in the glucose content were found in embryos of some teleost fishes; however, the origin of this glucose has been unknown. Unveiling new metabolic strategies in fish embryos has a potential for better aquaculture technologies. In the present study, using zebrafish, we assessed how these embryos obtain the glucose. We employed stable isotope (13C)-labeled substrates and injected them to the zebrafish embryos. Our liquid chromatography-mass spectrometry-based isotope tracking revealed that among all tested substrate, glutamate was most actively metabolized to produce glucose in the zebrafish embryos. Expression analysis for gluconeogenic genes found that many of these were expressed in the yolk syncytial layer (YSL), an extraembryonic tissue found in teleost fishes. Generation 0 (G0) knockout of pck2, a gene encoding the key enzyme for gluconeogenesis from Krebs cycle intermediates, reduced gluconeogenesis from glutamate, suggesting that this gene is responsible for gluconeogenesis from glutamate in the zebrafish embryos. These results showed that teleost YSL undergoes gluconeogenesis, likely contributing to the glucose supplementation to the embryos with limited glucose source. Since many other animal lineages lack YSL, further comparative analysis will be interesting.

Single-polyp metabolomics for coral health assessment.

Coral reef ecosystems supported by environmentally sensitive reef-building corals face serious threats from human activities. Our understanding of these reef threats is hampered by the lack of sufficiently sensitive coral environmental impact assessment systems. In this study, we established a platform for metabolomic analysis at the single-coral-polyp level using state-of-the-art mass spectrometry (probe electrospray ionization/tandem mass spectrometry; PESI/MS/MS) capable of fine-scale analysis. We analyzed the impact of the organic UV filter, benzophenone (BP), which has a negative impact on corals. We also analyzed ammonium and nitrate samples, which affect the environmental sensitivity of coral-zooxanthella (Symbiodiniaceae) holobionts, to provide new insights into coral biology with a focus on metabolites. The method established in this study breaks new ground by combining PESI/MS/MS with a technique for coral polyps that can control the presence or absence of zooxanthellae in corals, enabling functions of zooxanthellae to be assessed on a polyp-by-polyp basis for the first time. This system will clarify biological mechanisms of corals and will become an important model system for environmental impact assessment using marine organisms.

Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.

Characterization of recombinant photoconverting green fluorescent Akanes.

Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.

Coastal ecological impacts from pumice rafts.

An explosive volcanic eruption occurred in the Ogasawara Islands on 13-15 August 2021, bringing unprecedented amounts of floating pumice to the coast of Okinawa Island in the Ryukyu Archipelago, 1300 km west of the volcano, approximately 2 months later. The coast of Okinawa Island, especially along the northern part, is home to many typical subtropical seascapes, including coral reefs and mangrove forests, so the possible impact of the large amount of pumice is attracting attention. Here, we report early evidence of ecosystem changes as a result of large-scale pumice stranding on coastal beaches, in estuaries and mangrove forests and passage across fringing coral reefs. Massive pumice drifts are major obstacles to fishing activities and ship traffic, but short and long-term changes in coastal ecosystems can also occur. The phenomena observed on Okinawa Island can be a preview of coastal impacts for the Kyushu, Shikoku, Honshu Islands, where pumice has subsequently washed ashore.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains.

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.

Leishmanicidal phenolic compounds derived from Dalbergia cultrata.

Leishmaniasis is a protozoan tropical infection that is estimated to be more than 0.3 million new cases occur annually worldwide. A novel phenolic compound, cultratin A (1), was isolated as a leishmanicidal constituent from the timber of Dalbergia cultrata, along with three known neoflavanoids (2, 3, 4), two benzofurans (5, 6), and two phenolic compounds (7, 8). Their structures were determined using spectral methods. Among them, a new compound (1) and 4-(S)-methoxydalbergione (2) showed effective leishmanicidal activities (IC50: 2.0 and 2.6 µM, respectively), while compound 8 showed moderate activity (IC50: 11 µM). The cytotoxicity of compounds 1 and 2 was also weaker than that of the other compounds.

Trivalent Iron Is Responsible for the Yellow Color Development in the Nacre of Akoya Pearl Oyster Shells.

The gold and cream colors of cultured Akoya pearls, as well as natural yellow nacre of pearl oyster shells, are thought to arise from intrinsic yellow pigments. While the isolation of the yellow pigments has been attempted using a large amount of gold pearls, the substance concerned is still unknown. We report here on the purification and characterization of yellow pigments from the nacre of Akoya pearl oyster shells. Two yellow components, YC1 and YC2, were isolated from the HCl-methanol (HCl-MeOH) extract from nacreous organic matrices obtained by decalcification of the shells with ethylenediaminetetraacetic acid (EDTA). Energy-dispersive X-ray and infrared spectroscopy analyses suggested that YC1 and YC2 precipitated under basic conditions are composed of Fe-containing inorganic and polyamide-containing organic compounds, respectively. YC1 solubilized under acidic conditions exhibited positive reactions to KSCN and K4[Fe(CN)6] reagents, showing the same ultraviolet-visible absorption spectrum as those of Fe(III)-containing compounds. In addition, X-ray absorption fine structure analysis supported the compound in the form of Fe(III). The total amount of Fe was approximately 2.6 times higher in the yellow than white nacre, and most Fe was fractionated into the EDTA-decalcifying and HCl-MeOH extracts. These results suggest that Fe(III) coordinated to EDTA-soluble and insoluble matrix compounds are mainly associated with yellow color development not only in the Akoya pearl oyster shells but also in the cultured Akoya pearls.

Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.

A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.

Phosphate Enrichment Hampers Development of Juvenile Acropora digitifera Coral by Inhibiting Skeleton Formation.

Coral reef degradation due to various local stresses, such as nutrient enrichment and terrestrial run-off into coastal waters, is an increasing global concern. Inorganic phosphates have been considered to possibly inhibit skeleton formation in corals. Despite many studies available on the effects of nutrients on corals, a clear consensus on how nutrients exert deteriorative effects on corals has not been established satisfactorily. In this study, we examined the effects of phosphates and nitrates on in vitro aragonite CaCO3 formation by using biogenic polyamines and in vivo aragonite formation in the skeleton of juvenile Acropora digitifera corals. We showed that the phosphates at similar concentrations clearly inhibited both in vitro and in vivo CaCO3 formation. In contrast, nitrates inhibited neither in vitro aragonite CaCO3 formation nor in vivo aragonite formation in juvenile coral skeleton. Furthermore, our findings showed that inhibition of coral skeleton formation was due to absorption of phosphate on the skeleton, which inorganically inhibited normal development of juvenile coral skeleton.

Effects of moderate thermal anomalies on Acropora corals around Sesoko Island, Okinawa.

Over the past several decades, coral reef ecosystems have experienced recurring bleaching events. These events were predominantly caused by thermal anomalies, which vary widely in terms of severity and spatio-temporal distribution. Acropora corals, highly prominent contributors to the structural complexity of Pacific coral reefs, are sensitive to thermal stress. Response of Acropora corals to extremely high temperature has been well documented. However, studies on the effects of moderately high temperature on Acropora corals are limited. In the summer of 2016, a moderate coral bleaching event due to moderately high temperature was observed around Sesoko Island, Okinawa, Japan. The objective of this study was to examine thermal tolerance patterns of Acropora corals, across reefs with low to moderate thermal exposure (degree heating weeks ~2-5°C week). Field surveys on permanent plots were conducted from October 2015 to April 2017 to compare the population dynamics of adult Acropora corals 6 months before and after the bleaching events around Sesoko Island. Variability in thermal stress response was driven primarily by the degree of thermal stress. Wave action and turbidity may have mediated the thermal stress. Tabular and digitate coral morphologies were the most tolerant and susceptible to thermal stress, respectively. Growth inhibition after bleaching was more pronounced in the larger digitate and corymbose coral morphologies. This study indicates that Acropora populations around Sesoko Island can tolerate short-term, moderate thermal challenges.

Atmospheric CO2 captured by biogenic polyamines is transferred as a possible substrate to Rubisco for the carboxylation reaction.

Biogenic polyamines are involved in a wide range of plant cellular processes, including cell division, morphogenesis and stress responses. However, the exact roles of biogenic polyamines are not well understood. We recently reported that biogenic polyamines that have multiple amino groups can react with CO2 and accelerate calcium carbonate formation in seawater. The ability of biogenic polyamines to capture atmospheric CO2 prompted us to examine their roles in photosynthesis. Here, we demonstrated that atmospheric CO2 captured by biogenic polyamines is a candidate substrate for the carboxylation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is an enzyme involved in the first major step of carbon fixation during photosynthesis, and that biogenic polyamines can accelerate the carboxylation reaction of this enzyme because of their specific affinity for CO2. Moreover, the results of our nuclear magnetic resonance (NMR) analysis showed that putrescine, which is the most common biogenic polyamine, reacts with atmospheric CO2 and promotes the formation of carbamate derivatives and bicarbonate in aqueous environments. A sufficient amount of CO2 is well known to be produced by carbonic anhydrase from bicarbonate in vivo. The present study indicates that CO2 would be also produced by the equilibrium reaction from carbonate produced by biogenic polyamines and would be used as a substrate of Rubisco, too. Our results may suggest a new photosynthetic research strategy that involves CO2-concentrating mechanisms and also possibly constitutes a potential tool for reducing atmospheric CO2 levels and, consequently, global warming.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp (Marsupenaeus japonicus) acclimated at different salinities.

Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.

Dynamic changes in the accumulation of metabolites in brackish water clam Corbicula japonica associated with alternation of salinity.

The brackish water clam Corbicula japonica inhabits rivers and brackish waters throughout Japan where the major fishing grounds in the Ibaraki Prefecture, Japan, are located at the Hinuma Lake and Hinuma River. Water salinity in the Lake Hinuma is low and stable due to the long distance from the Pacific Ocean, whereas that in the downstream of the river varies daily due to a strong effect of tidal waters. In the present study, we dissected the gill and foot muscle of brackish water clam collected from these areas, and subjected them to metabolome analysis by capillary electrophoresis-time-of-flight mass spectrometry. More than 200 metabolites including free amino acids, peptides and organic acids were identified, and their amounts from the foot muscle tend to be higher than those from the gill. The principal component analysis revealed that the amount of each metabolite was different among sampling areas and between the gill and foot muscle, whereas no apparent differences were observed between male and female specimens. When the metabolites in the female clam at high salinity were compared with those at low salinity, concentrations of ß-alanine, choline, γ-aminobutyric acid, ornithine and glycine betaine were found to be changed in association with salinity. We also compared various metabolites in relation to metabolic pathways, suggesting that many enzymes were involved in their changes depending on salinity.

Biogenic polyamines capture CO2 and accelerate extracellular bacterial CaCO3 formation.

Bacteria, including cyanobacteria, as well as some fungi, are known to deposit calcium carbonate (CaCO(3)) extracellularly in calcium-containing artificial medium. Despite extensive investigation, the mechanisms involved in extracellular formation of CaCO(3) by bacteria have remained unclear. The ability of synthetic amines to remove carbon dioxide (CO(2)) from natural gas led us to examine the role of biogenic polyamines in CaCO(3) deposition by bacteria. Here, we demonstrated that biogenic polyamines such as putrescine, spermidine, and spermine were able to react with atmospheric CO(2) and the resultant carbamate anion was characterized by using nuclear magnetic resonance (NMR) analysis. Biogenic polyamines accelerated the formation of CaCO(3), and we artificially synthesized the dumbbell-shaped calcites, which had the same form as observed with bacterial CaCO3 precipitates, under nonbacterial conditions by using polyamines. The reaction rate of calcification increased with temperature with an optimum of around 40 °C. Our observation suggests a novel scheme for CO(2) dissipation that could be a potential tool in reducing atmospheric CO(2) levels and, therefore, global warming.

New sulfoalkylresorcinol from marine-derived fungus, Zygosporium sp. KNC52.

A new sulfoalkylresorcinol (1) was isolated from the marine-derived fungus, Zygosporium sp. KNC52. The structure of 1 was elucidated by spectroscopic methods including MS and NMR, and the absolute stereochemistry was determined by the modified Mosher's method. Compound 1 inhibited FtsZ polymerization in vitro and exhibited weak antimicrobial activity against multi-drug-resistant bacteria.